JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1977, p. 387-389
Copyright © 1977 American Society for Microbiology
Vol. 5, No. 4 Printed in U.S.A.
Simplified Media for Isolating Neisseria gonorrhoeae E. H. SNG,* V. S. RAJAN, AND A. L. LIM Department of Pathology, Singapore General Hospital, Singapore 3, Republic of Singapore Received for publication 1 November 1976
Two simplified media for isolating Neisseria gonorrhoeae are described. They differ from the modified Thayer-Martin medium in the enrichment and antibiotics used for preparation. The enrichment for one, enrichment 4 medium, contains only four ingredients, and amphotericin B is used instead of nystatin. This medium is comparable to the modified Thayer-Martin medium for isolating N. gonorrhoeae. It is convenient to prepare and is only one-third the cost of ThayerMartin medium. It is a suitable alternative to the modified Thayer-Martin. The enrichment 5 medium is a hemoglobin-free version of enrichment 4 medium. It is somewhat more selective for contaminants but is also more inhibitory for N. gonorrhoeae. An important contribution to the control of gonorrhea was made by the introduction of selective medium (4). Since then, many modifications have been made, and the modified Thayer-Martin (TM) medium (3) is an improvement over previous formulas. Our laboratory has been using a modified version of this medium (1.3% agar instead of 2.0%) for 3 years, and it has given satisfactory results. A major problem in using this medium has been the cost, and we describe below two simplified media that reduce the cost considerably.
(i) 1 ml; antibiotic solution (ii), 1 ml; dextrose solution (25%), 10 ml. The GC agar base (with added agar) and hemoglobin solutions were prepared separately in 500 ml of distilled water. After autoclaving, they were cooled to 56°C and mixed. The premixed enrichment, antibiotic, and dextrose solutions were then added. The medium was then dispensed into petri dishes. The final pH of the medium is 7.2. (ii) E5 medium. E5 medium was similar to the E4 medium, except that the hemoglobin and E4 solutions were replaced by 1 ml of E5 solution. This solution contained the same ingredients as E4 solution in addition to 5 mg of ferric nitrate per ml. (iii) Modified TM medium. Modified TM medium was similar to the E4 medium, except that the enMATERIALS AND METHODS richment and antibiotic solutions were replaced by Preparation of media. (i) E4 medium. Enrich- 10 ml of IsoVitaleX, 10 ml of VCN (BBL), and 0.25 ment 4 (E4) solution contained the following ingre- ml of trimethoprim lactate (20 mg/ml) for each liter dients (per milliliter) in distilled water: L-cysteine of medium. The final concentration of trimethoprim HCl, 260 mg; L-glutamine, 100 mg; coenzyme 1, 4 lactate was 5 mg/liter of medium. mg; cocarboxylase, 2 mg (all from Sigma Chemical Evaluation of media. For the laboratory evaluaCo.). The solution was stored in 1-ml samples at tion of media, 40 strains of recently isolated Neis-70°C. The enrichment has been kept at this tem- seria gonorrhoeae were suspended in nutrient broth. perature for 5 months without deterioration. Both These strains were identified by colonial morpholE4 and enrichment 5 (E5) solutions are modifica- ogy on modified TM medium, oxidase reaction, tions of earlier formulations (5; C. E. Lankford, Gram stain microscopic examination, and sugar ferBacteriol. Proc., G40, p. 20, 1950). mentations. The suspensions were streaked on the Antibiotic solutions (i) and (ii) contained the fol- different media, and the plates were incubated at lowing (per milliliter) in distilled water: (i) vanco- 36°C in a carbon dioxide atmosphere, using candle mycin (Eli Lilly & Co.), 3 mg; colistin methane jars. The plates were examined after overnight and sulfonate sodium (Banyu), 7.5 mg; trimethoprim 48-h incubations. For the inoculum dilution test, 10lactate (Burroughs Wellcome), 5 mg; and (ii) am- fold dilutions were made on 20 strain suspensions. A photericin B (E. R. Squibb & Sons), 1 mg. The two loopful of organisms was taken from each dilution antibiotic solutions were prepared separately, since and inoculated on the test media. The media were a precipitate was formed when colistin methane sul- incubated as above. The maximum inoculum titer fonate sodium was mixed with amphotericin B. The for which growth was detected on each medium was solutions were stored in 1-ml samples at -70°C. recorded. In the field trial, cervical and rectal swabs The completed E4 medium contained the follow- were taken from 597 patients attending a venereal ing (per liter) in distilled water: GC agar base disease clinic and inoculated on the different media. (BBL), 36 g; agar (granulated, BBL), 3 g; hemoglo- The order in which the media were inoculated was bin (BBL), 10 g; E4 solution, 1 ml; antibiotic solution rotated every day. The plates were incubated as 387
388
SNG, RAJAN, AND LIM
above. The number of times N. gonorrhoeae or any contaminant was isolated on each plate was recorded. Media contamination was rated according to the number of plates that grew at least one colony of contaminant. Vancomycin selectivity test. E4 and E5 media were prepared without adding the usual antibiotic. To 15-ml samples of each medium vancomycin was added to give the following final concentrations: 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ,ug/ml. These were then poured into petri dishes and allowed to set. Suspensions in nutrient broth were prepared from four strains of Staphylococcus epidermidis isolated from clinical material. These were streaked on the media and incubated at 37°C overnight. The next day the minimal inhibitory concentrations of vancomycin for the bacteria were determined on the two media.
RESULTS
J. CLIN. MICROBIOL.
TABLE 2. Comparison of results on cervical and rectal specimens taken from 597 women No. of N. gonorrhoeae isolated
Medium
Modified TM E4 E5
No. of contami-
nants isolated(
77 78 76
313 (52) 329 (55) 200 (34)
TABLE 3. Growth of four S. epidermidis strainsoon media containing vancomycin No. of strains growing at vancomycin concn Mediuma 0
0.5
1.0
1.5
2.0
2.5
3.0
E4
4
4
4
4
0
0
0
E5
4
4
0
0
0
0
0
In the laboratory evaluation of media, the 40 a Media contained no antibiotics other than vanstrains of N. gonorrhoeae grew equally well on comycin in the amounts shown. all three media tested. In the inoculum dilution test (Table 1), the E4 medium was comparable to the modified TM medium. The maximum containing medium was 2.0 ,ug/ml, whereas in inoculum titers for 18 strains were identical for the hemoglobin-free medium it was 1.0 jig/ml. both media. For one strain a few colonies were DISCUSSION grown on E4 medium at a dilution of 10-7, The E4 solution differs from IsoVitaleX in whereas on modified TM it grew at a dilution of 10-6. On the other hand, another strain grew at containing only four ingredients instead of 10-6 on modified TM but at 10-5 on E4 medium. twelve. The concentrations of the labile coenThe E5 medium was fractionally less sensitive zyme I and cocarboxylase were increased. Bethan the modified TM. Two strains grew on it cause of high solubility of these ingredients 1 liter of medium can be prepared with 1 ml of the at one titer lower than on modified TM. In the field trial, N. gonorrhoeae was isolated enrichment. This made preparation and stor78 times from E4 medium and 77 times from age of the enrichment easy. The preparation of modified TM. The growth on both media was IsoVitaleX from its ingredients is tedious, and comparable (Table 2). On E5 medium N. gonor- the final volume of enrichment is 10 ml/liter of rhoeae was isolated 76 times, but it was noticed medium. Simplified enrichments have been that on a number of occasions the colonies were previously used for the preparation of nonselecsmaller and fewer. E4 medium had slightly tive media for the gonococcus (5; Lankford, Bacmore contaminants (55%), that modified TM teriol. Proc., G40, p. 20, 1950). Amphotericin B has been chosen instead of (52%), but E5 medium had the least contamination (34%). The contaminants were mostly nystatin because it has been shown to be a more effective antifungal agent (1). Although the angram-positive cocci. The vancomycin selectivity test (Table 3) tibiotic solutions are in two 1-ml samples per showed that the antibiotic was less effective in liter of medium, they occupy less storage space the presence of hemoglobin. The minimal in- than the commercial VCN preparation. Behibitory concentration of the antibiotic for the cause of the small volumes needed per liter of four S. epidermidis strains in the hemoglobin- medium, both enrichment and antibiotic solutions may be conveniently lyophilized by laboratory freeze-dryers. TABLE 1. Comparison of data for growth of 20 N. The E4 medium was as sensitive as the modigonorrhoeae strains fied TM medium in supporting the growth ofN. No. of strains grown with maximum gonorrhoeae from laboratory cultures and painoculum titer of: tients. Contamination was slightly increased, but this did not affect the isolation rate from 10-4 10-5 10-7 10-8 patients. The beneficial effect expected of amModified TM 5 7 7 1 photericin B (1) was not seen in this study. This E4 5 8 5 2 was because the contaminants were mainly E5 5 8 7 gram-positive cocci. Medium
VOL. 5, 1977
MEDIA FOR ISOLATING N. GONORRHOEAE
The E5 medium was found to be slightly more inhibitory than the modified TM medium. In the inoculum dilution test, two strains grew at one titer lower than with the modified TM. In the field trial, though the isolation rate was comparable, the colonies were occasionally smaller and fewer. The contamination on this medium was also less than that found on the hemoglobin-containing media. As the contaminants were mostly gram-positive cocci, it was felt that the hemoglobin might be binding some of the vancomycin. This was confirmed by the finding that in the presence of hemoglobin a higher concentration of vancomycin was required to inhibit the four strains of S. epidermidis. Hence, in the hemoglobin-free medium the efficacy of vancomycin was higher, and it inhibited more contaminants and some of the vancomycin-sensitive N. gonorrhoeae. In a study involving male patients (2), it was found that a hemoglobin-free medium (containing IsoVitaleX, vancomycin, colistin, and nystatin) also grew fewer contaminants than the TM medium. However, the isolation of N. gonorrhoeae was not adversely affected. This could be because the N. gonorrhoeae strains were more resistant to vancomycin than those in this
study. The cost of preparing the E4 medium is approximately one-third that of the modified TM medium. It is easy to prepare, and storage for the enrichment and antibiotic solutions takes up less space than the corresponding commercial preparations for the modified TM medium. It is a suitable alternative to the modified TM medium for isolating N. gonorrhoeae from pa-
389
tients. The presence of asymptomatic carriers ofN. gonorrhoeae in both males and females is now well recognized. A comprehensive program to detect this vast reservoir of asymptomatic carriers might involve extending the screening program for gonorrhea to lower-risk groups. The availability of a low-cost medium, such as the E4 medium, should make this more feasible economically. The E5 medium is even cheaper. It is also more convenient to produce, as there is no necessity to prepare the hemoglobin solution. Although the isolation rate for N. gonorrhoeae on the E5 medium is comparable to that on the modified TM, it is fractionally more inhibitory. Further study is being carried out to make it less inhibitory. ACKNOWLEDGMENT This study was supported by a grant from the World Health Organization. LITERATURE CITED 1. Faur, Y. C., M. H. Weisburd, and M. E. Wilson. 1973. A new medium for the isolation of pathogenic Neisseria (N.Y.C. medium). II. Effect of amphotericin B and trimethoprim lactate on selectivity. Health Lab. Sci. 10:55-60. 2. Jacobs, N. F., Jr., and S. J. Kraus. 1975. Comparison of hemoglobin-free culture media and Thayer-Martin medium for the primary isolation of Neisseria gonorrhoeae. J. Clin. Microbiol. 1:401-404. 3. Martin, J. E., J. H. Armstrong, and P. B. Smith. 1974. New system for cultivation of Neisseria gonorrhoeae. Appl. Microbiol. 27:802-805. 4. Thayer, J. D., and J. E. Martin. 1964. A selective medium for the cultivation of N. gonorrhoeae and N. meningitidis. Public Health Rep. 79:49-67. 5. White, L. A., and D. S. Kellogg, Jr. 1965. An improved fermentation medium for Neisseria gonorrhoeae and other Neisseria. Health Lab. Sci. 2:238-241.
Copyright © 1977 American Society for Microbiology
Vol. 5, No. 4 Printed in U.S.A.
Simplified Media for Isolating Neisseria gonorrhoeae E. H. SNG,* V. S. RAJAN, AND A. L. LIM Department of Pathology, Singapore General Hospital, Singapore 3, Republic of Singapore Received for publication 1 November 1976
Two simplified media for isolating Neisseria gonorrhoeae are described. They differ from the modified Thayer-Martin medium in the enrichment and antibiotics used for preparation. The enrichment for one, enrichment 4 medium, contains only four ingredients, and amphotericin B is used instead of nystatin. This medium is comparable to the modified Thayer-Martin medium for isolating N. gonorrhoeae. It is convenient to prepare and is only one-third the cost of ThayerMartin medium. It is a suitable alternative to the modified Thayer-Martin. The enrichment 5 medium is a hemoglobin-free version of enrichment 4 medium. It is somewhat more selective for contaminants but is also more inhibitory for N. gonorrhoeae. An important contribution to the control of gonorrhea was made by the introduction of selective medium (4). Since then, many modifications have been made, and the modified Thayer-Martin (TM) medium (3) is an improvement over previous formulas. Our laboratory has been using a modified version of this medium (1.3% agar instead of 2.0%) for 3 years, and it has given satisfactory results. A major problem in using this medium has been the cost, and we describe below two simplified media that reduce the cost considerably.
(i) 1 ml; antibiotic solution (ii), 1 ml; dextrose solution (25%), 10 ml. The GC agar base (with added agar) and hemoglobin solutions were prepared separately in 500 ml of distilled water. After autoclaving, they were cooled to 56°C and mixed. The premixed enrichment, antibiotic, and dextrose solutions were then added. The medium was then dispensed into petri dishes. The final pH of the medium is 7.2. (ii) E5 medium. E5 medium was similar to the E4 medium, except that the hemoglobin and E4 solutions were replaced by 1 ml of E5 solution. This solution contained the same ingredients as E4 solution in addition to 5 mg of ferric nitrate per ml. (iii) Modified TM medium. Modified TM medium was similar to the E4 medium, except that the enMATERIALS AND METHODS richment and antibiotic solutions were replaced by Preparation of media. (i) E4 medium. Enrich- 10 ml of IsoVitaleX, 10 ml of VCN (BBL), and 0.25 ment 4 (E4) solution contained the following ingre- ml of trimethoprim lactate (20 mg/ml) for each liter dients (per milliliter) in distilled water: L-cysteine of medium. The final concentration of trimethoprim HCl, 260 mg; L-glutamine, 100 mg; coenzyme 1, 4 lactate was 5 mg/liter of medium. mg; cocarboxylase, 2 mg (all from Sigma Chemical Evaluation of media. For the laboratory evaluaCo.). The solution was stored in 1-ml samples at tion of media, 40 strains of recently isolated Neis-70°C. The enrichment has been kept at this tem- seria gonorrhoeae were suspended in nutrient broth. perature for 5 months without deterioration. Both These strains were identified by colonial morpholE4 and enrichment 5 (E5) solutions are modifica- ogy on modified TM medium, oxidase reaction, tions of earlier formulations (5; C. E. Lankford, Gram stain microscopic examination, and sugar ferBacteriol. Proc., G40, p. 20, 1950). mentations. The suspensions were streaked on the Antibiotic solutions (i) and (ii) contained the fol- different media, and the plates were incubated at lowing (per milliliter) in distilled water: (i) vanco- 36°C in a carbon dioxide atmosphere, using candle mycin (Eli Lilly & Co.), 3 mg; colistin methane jars. The plates were examined after overnight and sulfonate sodium (Banyu), 7.5 mg; trimethoprim 48-h incubations. For the inoculum dilution test, 10lactate (Burroughs Wellcome), 5 mg; and (ii) am- fold dilutions were made on 20 strain suspensions. A photericin B (E. R. Squibb & Sons), 1 mg. The two loopful of organisms was taken from each dilution antibiotic solutions were prepared separately, since and inoculated on the test media. The media were a precipitate was formed when colistin methane sul- incubated as above. The maximum inoculum titer fonate sodium was mixed with amphotericin B. The for which growth was detected on each medium was solutions were stored in 1-ml samples at -70°C. recorded. In the field trial, cervical and rectal swabs The completed E4 medium contained the follow- were taken from 597 patients attending a venereal ing (per liter) in distilled water: GC agar base disease clinic and inoculated on the different media. (BBL), 36 g; agar (granulated, BBL), 3 g; hemoglo- The order in which the media were inoculated was bin (BBL), 10 g; E4 solution, 1 ml; antibiotic solution rotated every day. The plates were incubated as 387
388
SNG, RAJAN, AND LIM
above. The number of times N. gonorrhoeae or any contaminant was isolated on each plate was recorded. Media contamination was rated according to the number of plates that grew at least one colony of contaminant. Vancomycin selectivity test. E4 and E5 media were prepared without adding the usual antibiotic. To 15-ml samples of each medium vancomycin was added to give the following final concentrations: 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ,ug/ml. These were then poured into petri dishes and allowed to set. Suspensions in nutrient broth were prepared from four strains of Staphylococcus epidermidis isolated from clinical material. These were streaked on the media and incubated at 37°C overnight. The next day the minimal inhibitory concentrations of vancomycin for the bacteria were determined on the two media.
RESULTS
J. CLIN. MICROBIOL.
TABLE 2. Comparison of results on cervical and rectal specimens taken from 597 women No. of N. gonorrhoeae isolated
Medium
Modified TM E4 E5
No. of contami-
nants isolated(
77 78 76
313 (52) 329 (55) 200 (34)
TABLE 3. Growth of four S. epidermidis strainsoon media containing vancomycin No. of strains growing at vancomycin concn Mediuma 0
0.5
1.0
1.5
2.0
2.5
3.0
E4
4
4
4
4
0
0
0
E5
4
4
0
0
0
0
0
In the laboratory evaluation of media, the 40 a Media contained no antibiotics other than vanstrains of N. gonorrhoeae grew equally well on comycin in the amounts shown. all three media tested. In the inoculum dilution test (Table 1), the E4 medium was comparable to the modified TM medium. The maximum containing medium was 2.0 ,ug/ml, whereas in inoculum titers for 18 strains were identical for the hemoglobin-free medium it was 1.0 jig/ml. both media. For one strain a few colonies were DISCUSSION grown on E4 medium at a dilution of 10-7, The E4 solution differs from IsoVitaleX in whereas on modified TM it grew at a dilution of 10-6. On the other hand, another strain grew at containing only four ingredients instead of 10-6 on modified TM but at 10-5 on E4 medium. twelve. The concentrations of the labile coenThe E5 medium was fractionally less sensitive zyme I and cocarboxylase were increased. Bethan the modified TM. Two strains grew on it cause of high solubility of these ingredients 1 liter of medium can be prepared with 1 ml of the at one titer lower than on modified TM. In the field trial, N. gonorrhoeae was isolated enrichment. This made preparation and stor78 times from E4 medium and 77 times from age of the enrichment easy. The preparation of modified TM. The growth on both media was IsoVitaleX from its ingredients is tedious, and comparable (Table 2). On E5 medium N. gonor- the final volume of enrichment is 10 ml/liter of rhoeae was isolated 76 times, but it was noticed medium. Simplified enrichments have been that on a number of occasions the colonies were previously used for the preparation of nonselecsmaller and fewer. E4 medium had slightly tive media for the gonococcus (5; Lankford, Bacmore contaminants (55%), that modified TM teriol. Proc., G40, p. 20, 1950). Amphotericin B has been chosen instead of (52%), but E5 medium had the least contamination (34%). The contaminants were mostly nystatin because it has been shown to be a more effective antifungal agent (1). Although the angram-positive cocci. The vancomycin selectivity test (Table 3) tibiotic solutions are in two 1-ml samples per showed that the antibiotic was less effective in liter of medium, they occupy less storage space the presence of hemoglobin. The minimal in- than the commercial VCN preparation. Behibitory concentration of the antibiotic for the cause of the small volumes needed per liter of four S. epidermidis strains in the hemoglobin- medium, both enrichment and antibiotic solutions may be conveniently lyophilized by laboratory freeze-dryers. TABLE 1. Comparison of data for growth of 20 N. The E4 medium was as sensitive as the modigonorrhoeae strains fied TM medium in supporting the growth ofN. No. of strains grown with maximum gonorrhoeae from laboratory cultures and painoculum titer of: tients. Contamination was slightly increased, but this did not affect the isolation rate from 10-4 10-5 10-7 10-8 patients. The beneficial effect expected of amModified TM 5 7 7 1 photericin B (1) was not seen in this study. This E4 5 8 5 2 was because the contaminants were mainly E5 5 8 7 gram-positive cocci. Medium
VOL. 5, 1977
MEDIA FOR ISOLATING N. GONORRHOEAE
The E5 medium was found to be slightly more inhibitory than the modified TM medium. In the inoculum dilution test, two strains grew at one titer lower than with the modified TM. In the field trial, though the isolation rate was comparable, the colonies were occasionally smaller and fewer. The contamination on this medium was also less than that found on the hemoglobin-containing media. As the contaminants were mostly gram-positive cocci, it was felt that the hemoglobin might be binding some of the vancomycin. This was confirmed by the finding that in the presence of hemoglobin a higher concentration of vancomycin was required to inhibit the four strains of S. epidermidis. Hence, in the hemoglobin-free medium the efficacy of vancomycin was higher, and it inhibited more contaminants and some of the vancomycin-sensitive N. gonorrhoeae. In a study involving male patients (2), it was found that a hemoglobin-free medium (containing IsoVitaleX, vancomycin, colistin, and nystatin) also grew fewer contaminants than the TM medium. However, the isolation of N. gonorrhoeae was not adversely affected. This could be because the N. gonorrhoeae strains were more resistant to vancomycin than those in this
study. The cost of preparing the E4 medium is approximately one-third that of the modified TM medium. It is easy to prepare, and storage for the enrichment and antibiotic solutions takes up less space than the corresponding commercial preparations for the modified TM medium. It is a suitable alternative to the modified TM medium for isolating N. gonorrhoeae from pa-
389
tients. The presence of asymptomatic carriers ofN. gonorrhoeae in both males and females is now well recognized. A comprehensive program to detect this vast reservoir of asymptomatic carriers might involve extending the screening program for gonorrhea to lower-risk groups. The availability of a low-cost medium, such as the E4 medium, should make this more feasible economically. The E5 medium is even cheaper. It is also more convenient to produce, as there is no necessity to prepare the hemoglobin solution. Although the isolation rate for N. gonorrhoeae on the E5 medium is comparable to that on the modified TM, it is fractionally more inhibitory. Further study is being carried out to make it less inhibitory. ACKNOWLEDGMENT This study was supported by a grant from the World Health Organization. LITERATURE CITED 1. Faur, Y. C., M. H. Weisburd, and M. E. Wilson. 1973. A new medium for the isolation of pathogenic Neisseria (N.Y.C. medium). II. Effect of amphotericin B and trimethoprim lactate on selectivity. Health Lab. Sci. 10:55-60. 2. Jacobs, N. F., Jr., and S. J. Kraus. 1975. Comparison of hemoglobin-free culture media and Thayer-Martin medium for the primary isolation of Neisseria gonorrhoeae. J. Clin. Microbiol. 1:401-404. 3. Martin, J. E., J. H. Armstrong, and P. B. Smith. 1974. New system for cultivation of Neisseria gonorrhoeae. Appl. Microbiol. 27:802-805. 4. Thayer, J. D., and J. E. Martin. 1964. A selective medium for the cultivation of N. gonorrhoeae and N. meningitidis. Public Health Rep. 79:49-67. 5. White, L. A., and D. S. Kellogg, Jr. 1965. An improved fermentation medium for Neisseria gonorrhoeae and other Neisseria. Health Lab. Sci. 2:238-241.
